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Biochimica et Biophysica Acta 1793 (2009) 1110–1114

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Biochimica et Biophysica Acta

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Expression profiles of three novel sensory guanylate and guanylate -activating in the zebrafish retina

Nina Rätscho, Alexander Scholten, Karl-Wilhelm Koch ⁎

Biochemistry Group, Institute of Biology and Environmental Science, Faculty V, Carl von Ossietzky University Oldenburg, D-26111 Oldenburg, Germany

article info abstract

Article history: Three membrane bound sensory guanylate cyclases are expressed in photoreceptor cells of the developing Received 30 September 2008 and adult zebrafish retina. First appearance of mRNA transcripts was detected by in situ hybridization Received in revised form 19 November 2008 techniques for all guanylate cyclases between 3 and 4 days post fertilization (dpf), but only one isoform Accepted 27 December 2008 ( 3) appeared to be specifically expressed in cones of the adult retina. Transcripts of three Available online 6 January 2009 cone specific guanylate cyclase-activating proteins (zGCAP3, zGCAP4 and zGCAP7) were also detected at 3–

Keywords: 4 dpf. The expression onset of the guanylate cyclases and these neuronal calcium sensor proteins mainly Guanylate cyclase overlapped. High guanylate cyclase activities in larval eye preparations and the precisely controlled GCAP coexpression of guanylate cyclases and zGCAPs coincide with the onset of visual function at 3–4 dpf. Neuronal calcium sensor © 2009 Elsevier B.V. All rights reserved. Retina

1. Introduction of the full-length sequences and a characterization of their molecular properties has not been reported so far. Vertebrate rod and cone photoreceptor cells respond to light by a On the molecular level excitation and adaptation in cones are less change of the membrane potential. A light-triggered enzymatic well understood than in rods. But recently, the zebrafish has attained cascade leads to the hydrolysis of the intracellular messenger increasing interest as a model organism to study cone vision. guanosine-3′,5′-cyclic monophospate (cGMP), which is synthesized Zebrafish larvae respond to visual stimuli as early as 3 days post by membrane bound guanylate cyclases [1]. While rod cells operate at fertilization (3 dpf) and electroretinogram (ERG) recordings indicate low light intensities, cone cells are sensitive at higher levels of retinal function in larvae at 4 dpf (see for example [13–16]). The ambient illumination and maintain their responsiveness at fluctuating visual system of the zebrafish contains five types: background light [2]. Adaptation to different background light rods, double cones consisting of a long and a short member (DC), intensities is mediated by an operative network of photoreceptor long single cones (LSC) and short single cones (SSC). These five cell proteins and a change of intracellular calcium concentration. Calcium types express a total of nine opsin (one rod opsin and eigth sensor proteins detect these changes and regulate their targets in a cone opsin genes). The absorption characteristics of the opsins in calcium dependent manner [1–6]. Among them are the recoverins and cone cells indicate that zebrafish posses colour vision similar to that the guanylate cyclase-activating proteins (GCAPs) [3–5] that belong to of human [17,18]. the family of neuronal calcium sensor proteins [6,7]. Mammalian The large variety of GCAPs in the zebrafish retina points to a GCAP1 and GCAP2 are expressed in rods and cones and regulate complex calcium-sensing regulation of membrane-bound GCs. Thus, membrane bound guanylate cyclases in a calcium-dependent way, but in the present study we investigated if the putative sensory zGCs are differ in their biochemical and physiological properties [8–10]. expressed in photoreceptors of the adult zebrafish retina and whether A larger variety of GCAPs was recently detected in retina of adult they show a temporal and spatial overlapping coexpression pattern zebrafish [11], among them are four isoforms (zGCAP3, zGCAP4, with one or more cone specific GCAPs in the larvae. zGCAP5 and zGCAP7) specifically expressed in cones [11,12]. However, almost nothing is known about guanylate cyclases in the zebrafish 2. Materials and methods retina as possible targets of zGCAPs. Only three mRNA sequences that code for putative sensory guanylate cyclases (zGCs) had been 2.1. Fish maintenance deposited in the GenBank database (gucy2f, gc2, gc3), but the cloning All experiments on zebrafish were performed in accordance with the European Communities Council Directive for animal use in science – ⁎ Corresponding author. Tel.: +49 441 798 3640; fax: +49 441 798 193640. (86/609/EEC). Fish were kept in a 14:10 h day night cycle at either E-mail address: [email protected] (K.-W. Koch). 25 °C (adult) or 28.5 °C (larvae) according to standard conditions.

0167-4889/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2008.12.021 N. Rätscho et al. / Biochimica et Biophysica Acta 1793 (2009) 1110–1114 1111

2.2. Preparations of larvae for whole-mount in situ hybridization sequence of gc3 was also updated (XM_690001.3), but we could not confirm this sequence information. Instead, our sequence Larvae were raised in 0.003% 1-phenyl-2-thiourea (Sigma) to information obtained by cloning strategies confirmed the sequence prevent pigmentation. If necessary chorions were removed, larvae deposited under XM_690001.2. were anesthetized by chilling on ice and immediately fixed in 4% paraformaldehyde (PFA)/1×PBS (137 mM NaCl, 2.7 mM KCl, 8 mM 2.5. In situ hybridization

NaH2PO4, 2 mM KH2PO4, pH 7.4). After fixation larvae were washed in 1×PBS, dehydrated in methanol and stored at −20 °C. The 3′UTR containing regions of zGCAPs cDNA were amplified from a retinal cDNA library (library 760, Deutsches Ressourcenzentrum für 2.3. Preparation of adult retina Genomforschung GmbH, Berlin; used for zGCAP3, zGCAP4) or by cDNA first strand preparations from adult retina (zGCAP5, zGCAP7) Adult zebrafishes were anesthetized with tricaine and decapitated. using specific primer pairs (for zGCAP4 and zGCAP7 primer pairs Eyecups were fixed in 4% PFA/1×PBS for 20 min, cryoprotected by see [11]; for zGCAP3 and zGCAP5 see Table 1 in Supplementary bathing in 30% sucrose/1×PBS overnight at 4 °C and embedded with material). Amplified fragments were cloned into the pGEM-T-Easy liquid nitrogen in 33% Tissue Tek® (Sakura Finetech)/20% sucrose/ Vector (Promega), linearized with suitable restriction and 1×PBS and stored at −80 °C. Cryoembedded tissue was sectioned in processed for in vitro transcription of riboprobes using digoxigenin- 8–15 μm slices with a cryostat. labeled uracil (Roche). Whole-mount in situ hybridization procedure of larvae was done 2.4. In silico analyses of membrane bound retinal zebrafish guanylate as described [22]. In situ hybridization of cryosections was done cyclases and cloning of partial zGC fragments according to standard protocols [12] with slight modifications.

Putative sensory guanylate cyclases were identified by two 2.6. Guanylate cyclase assay independent data base searches using the BLAST algorithm [19]:In a first step we used the partial cDNA sequences RET-GC1 (GenBank Eyes of zebrafish larvae were enucleated at 10 dpf in Ringer accession number AY050503), RET-GC2 (AY050504), and RET-GC3 solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2 and 5 mM HEPES (AY050505) published in [20] to screen the GenBank data base. pH 7.4), immediately frozen in liquid nitrogen and stored at −80 °C. The second search was done in the Danio rerio RefSeq RNA-data After thawing a buffer containing 20 mM HEPES pH 7.4, 120 mM KCl, base using the sequences for retina specific sensory guanylate 5 mM MgCl2, 1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl cyclases from medaka (OlGC3: GenBank accession number fluoride was added to the eyes. The suspension was homogenized and AB000899; OlGC4: AB000900; OlGC5: AB000901; OlGC-R2: used for each determination of GC activity [23]. Incubation time was AB015874; see [21]) as query. By this strategy we obtained three 30 min at 30 °C. Protein content was determined by the amido black predicted mRNA sequences for zebrafish sensory guanylate method [24]. cyclases: XM_864538.1 with a length of 3324 bp corresponds to gucy2f [20] and is the ortholog to medakafish OlGC4; XM_683407.2 3. Results (3324 bp) contains the sequence gc2 and is comparable to OlGC- R2; XM_690001.2 (3688 bp) codes for gc3 and is similar to OlGC3. 3.1. Sensory zGCs are expressed in larval and adult eyes PCR strategies were used to ensure that the predicted sequences are expressed in the zebrafish retina. For each zebrafish Zebrafish larval and adult eyes express functional GCs. In order to GC-isoform we amplified overlapping cDNA fragments using test for zGC activities we prepared whole eyes from larvae at 10 dpf, reverse transcribed RNA from adult retina as template. Primers homogenized them and measured GC activities of these preparations. designed for this PCR strategy are listed in Table 1 (Supplementary The homogenates showed rather high GC activities at 6–8 nmol cGMP material). The amplified cDNA fragments were cloned in appro- synthesized/min and mg protein. Similar results were obtained with priate cloning vectors and verified by DNA sequencing. For gucy2f adult eye preparations. Recently, we were able to show that guanylate and gc2 the confirmed sequences were recently updated cyclases in membranes of adult zebrafish retina can be activated by (NM_131864.2, gucy2f and NM_001109695.1, gc2). The predicted recombinant zGCAP4 [25].

Fig. 1. Comparison of amino acid sequences of zGC3 with human ROS-GC2 (A) and human GC-A (B). Analysis was performed with the program PDOTPLOT, version 3.13 (Dr. W. Bönigk, INB-1, FZ-Jülich, Germany) using the following parameter: matrix MDM [30], window 12 amino acids, cutoff score 300. The gray bars on top indicate the extracellular and the black bars the intracellular domains of the GCs. The gene bank accession numbers were NM_000906 for hGC-A and L37378 for human hGC-2. 1112 N. Rätscho et al. / Biochimica et Biophysica Acta 1793 (2009) 1110–1114

comparison with known membrane bound GCs showed that all three zebrafish GC isoforms resembled sensory GCs and were different from receptor GCs that are controlled by external hormone ligands [26]. The three zGCs showed the highest degree of relationship to ROS-GC2 (retGC2, GC-F) that is expressed in mammalian retina [27,28]. The sequence comparison of zGC3 with the human receptor GC-A and ROS-GC2 is exemplified in Fig. 1A and B in a dot blot matrix analysis highlighting homologous and different regions. A high degree of is recognized as a diagonal black line. The almost complete lack of sequence homology is apparent in the extracellular domains of human GC-A and zGC3 (Fig. 1B).

3.2. ZGC3 displays cone specific expression in the adult zebrafish retina

Antisense RNA derived from the putative cyclase catalytic domains of zGC1, zGC2 and zGC3 were used for in situ hybridization studies and revealed specific transcription of all three GC isoforms in the adult retina. Signals were restricted to the outer nuclear layer (ONL), where the cell bodies of the photoreceptor cells are located. Transcripts of zGC3 were seen in DC, LSC and SSC (Fig. 2). A different expression profile was seen with the zGC1 and zGC2 antisense probes as they mainly labelled cell bodies of rods (RN) and SSC (Fig. 2). No staining was observed with the sense control probes (Fig. 2, right panel). These results demonstrated that zGC1, zGC2 and zGC3 are expressed in photoreceptor cells and that zGC3 appears as a cone specific GC. Furthermore, we here show for the first time that the predicted zGC sequences deposited in the data base (s. 2.4. in the Methods section) represent transcribed genes of the zebrafish retina.

3.3. Expression of zGC isoforms in the larval retina

Since the expression of zGCs in the adult zebrafish retina was photoreceptor specific we hypothesized that the expression proceeds during the retina maturation period when the visual system becomes functional. Therefore we investigated the developmental time course of zGC Fig. 2. Localization of zGC-transcripts in the adult zebrafish retina by in situ appearance in zebrafish larvae by in situ hybridization using zGC hybridization. Cryosections of the adult retina were treated with the antisense and antisense RNA probes. Transcripts were detected between 3–4 dpf for control sense probe. Prominent signals of zGC3 transcripts were detected in the ONL zGC1, zGC2 and zGC3 in the retina, as an example the detection of with staining of SSC (closed arrowhead), DC (bold arrow) and LSC (thin arrrow). Faint zGC3 transcripts is shown (Fig. 3, left part). However, the spatial signals in cones were observed with zGC1 and zGC2 transcripts. Rod nuclei (RN; open arrowhead) were only stained by antisense probes of zGC1 and zGC2. Scale bar: 20 μm. expression pattern differed between the three GC forms. Transcripts of zGC3 were first detected between 3–3.5 dpf in distinct clusters of photoreceptor cells starting in a ventral to central position (Figs. 3 and A combination of in silico analysis and cloning of overlapping cDNA 4) in the ONL. After 3.5 dpf the zGC3 signal expanded through the fragments revealed the nucleic acid sequences that code for three whole ONL, with less intense labelling of the ventral region. The membrane bound GCs, two with a length of 1107 amino acids and one outermost periphery of the photoreceptor layer was free of staining. of 1137 amino acids. These GCs were named zGC1, zGC2 and zGC3 The corresponding sense probe showed no staining (Fig. 4). The zGC1 corresponding to the genes gucy2f, gc2 and gc3, respectively. A probe labeled a cluster of photoreceptor cells in ventral position at 3–

Fig. 3. Localization of zGC3 and zGCAP3 transcripts in the larval zebrafish retina. Transcripts were localized in zebrafish larvae by whole-mount in situ hybridization (sense and antisense) at indicated time (3–4 dpf). Black arrows point to location of early stained photoreceptor cell clusters. The white arrows point to the corresponding regions in control larvae. Scale bars: 50 μm. N. Rätscho et al. / Biochimica et Biophysica Acta 1793 (2009) 1110–1114 1113

Fig. 4. Distribution of zGCAP and zGC transcripts in the larval zebrafish retina. Sections of whole-mount in situ hybridized larvae were analyzed by light microscopy. Developmental stage of larvae is indicated at the top of each figure. Black arrows point to locations of hybridization signal. Specific labelling of the ONL is seen as following: hybridization signals of zGC3 and zGCAP3 transcripts extended to the whole layer; zGC1 antisense probe labeled a ventrally located cluster of cells; hybridization signal of zGCAP4 was detected in a dorsal to central region. The white arrows point to the corresponding regions of the sense control. Scale bars: 50 μm.

3.5 dpf (Fig. 4); zGC2 showed the same spatial expression pattern, but 3 dpf and electroretinogram recordings indicate retinal function in the signal appeared later at 3.5–4 dpf (not shown). larvae at 4 dpf (see for example [13–16]). Thus, expression of zGCs coincided with the onset of visual function at 3–4 dpf. Further, three 3.4. Transcriptional onset of cone specific zGCAP genes cone specific zGCAPs were also expressed at the same stage of retina maturation. The cone specific expression of zGC3 led us ask, which of the cone Spatial localization of zGC and zGCAP transcripts by in situ specific zGCAPs exhibit a similar spatial–temporal expression pattern. hybridization was detected in the ONL. No transcripts were detected Whole-mount in situ hybridization of zebrafish larvae showed in the outermost periphery of the photoreceptor cell layer adjacent to appearance of zGCAP3 transcripts in the retina at the beginning of the marginal zone. This region is described as the area of cone genesis day 3 (Fig. 3) in spatially restricted clusters of photoreceptor cells, and maturation [29], which further indicates that zGCs and zGCAPs starting first in ventral position (Fig. 4) and a few hours later in central are mainly expressed in mature and functional cones. position. Among all zGC and zGCAP isoforms hybridization signals of However, among the three zGCs only zGC3 appeared cone specific, zGCAP3 and zGC3 transcripts showed the highest degree of overlap. since zGC1 and zGC2 were also detected in rods of the adult retina. The Onset of zGCAP4 transcription was detected between 3.5 dpf and 4 dpf strong expression of zGC3 in DC and LSC correlated with the (Fig. 4). The hybridization signal was observed in the ONL from the expression of all cone specific zGCAP isoforms. Thus, zGC3 might dorsal to central region, which is contrary to the localization of zGC3 not exclusively be regulated by one zGCAP form; instead it would and zGCAP3 transcripts. Photoreceptor cells in the dorsal and ventral represent a prime candidate to form regulator-target complexes with periphery of ONL were free of staining (Fig. 4). A similar transcription several zGCAPs in cones. pattern was observed with the zGCAP7 probe (not shown). The first appearance of zGCAP5 transcripts was detected rather late in larval Acknowledgement development around 15 dpf. All hybridization studies were repeated at least 8 times with similar results. We thank Dr. S. Rinkwitz for helpful discussion in the starting phase of the project. This work was supported by a grant form the Deutsche Forschungsgemeinschaft (Ko 948/7-1). 4. Discussion

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